以系统学为基础的蔷薇科DNA条形码研究

DNA barcoding is a technique which uses DNA sequence information to identify specimens. It was put on the stage of life science by increasingly demands for both the sciences and the society that quick and mass species identifications are required. It has been made possible today for construction of large DNA sequence reference libraries due to constantly reduced costs on DNA sequencing. ..DNA barcoding was once criticized for ignoring the species concept and thus questioned for its applicability. The works so far took the species described by taxonomists for grant because speices problem is not a target of DNA barcoding. But species does matter in DNA barcoding and DNA barcoding should not divorce itself from taxonomy and molecular systematics. DNA barcoding originated from DNA taxonomy and are aimed at species identification, the work taxonomy and molecular systematics do. The real situations of species are much more complex than the DNA barcoders had imagined. For the interrelated aims that taxonomy is to describe true species, molecular systematics is to establish the phylogenetic relationships of species, and DNA barcoding is to correctly identify species, the involvements of taxonomists and molecular systematists in DNA barcoding are absolutely essential for the success of DNA barcoding and it is time to link them together and to build DNA barcoding on the correct species delimitations. ..The aim of the project is, choosing economically important and technically practicable family, Rosaceae, as an example, to exemplify the necessity and feasibility of building DNA barcoding on reliable species delimitations through the taxonomic and molecular systematic work. More specifically, in addition to the suggested universal candidate barcodes, we are going to (1) develop better molecular markers to resolve the phylogenetic relationships above the species ranks within Rosaceae, which is the framework for next studies; (2) verify the species within Amygdaloideae based both morphological and molecular data and re-delimitate them if necessary; and (3) establish a DNA sequence reference library for unambiguous identifications of the species in Amygdaloideae. By carrying out the above mentioned work, we may meet the obstacles such as incorrect identification by chloroplast markers, rapid speciation through radiation, hybridization, polyploidization, apomixis and incomplete lineage sorting, etc. and ways for overcoming them thence would be found.It is expected that this project will serve as an example for DNA barcoding in the early stage of reference sequence library construction.

DNA条形码技术简单地说就是利用物种特有的DNA序列信息鉴别物种。DNA条形码技术的应用是科学技术发展和学科交叉的必然结果，是分类学广泛应用的必由之路，是基础研究成果转化为服务社会需求的典型案例。.DNA条形码技术曾备受质疑：DNA条形码技术真的能鉴别物种吗？这其实是个关于物种的科学问题，不是DNA条形码技术所能回答的，而必须由分类学和分子系统学来回答。因此，DNA条形码技术必须与分类学和分子系统学有机结合起来。因此，本项目拟以具有重要经济意义和深厚研究基础的蔷薇科为研究对象，在通用条形码的基础上，开发更高分辨率的标记，用系统发育的方法审定物种的范畴，探讨DNA条形码技术与分类学和分子系统学有机结合的必要性，为DNA条形码技术应用中可能遇到的叶绿体基因标记不可靠、快速物种分化、杂交、多倍化、无融合生殖"小种"、不完全谱系分选问题提供解决的成功案例，促进我国DNA条形码技术健康发展。

本项目采用分工协作、各司其职，相互配合、发挥专长的运作模式，圆满完成了各项研究内容，取得了丰硕的成果：发表了影响因子3以上的SCI研究论文7篇和发明专利1项；两度参加International Barcode of Life Conference并组织分会；培养了两名科研骨干、一名博士、两名博士生和两名硕士毕业生；为丰台工商分局、怀柔食药监局和广东迪艾生光电技术有限公司提供了食品添加物的原植物物种鉴定服务。主要研究成果总结如下。.1..开发了高分辨率叶绿体基因ycf1和单拷贝核基因Sbe1。.为了解决用现有植物DNA条形码分辨率低的问题，我们测定了大量的叶绿体基因组。通过比较叶绿体全基因组学研究，找到了位于ycf1基因内的高变区域，设计出适合的通用引物。经检验ycf1引物的扩增成功率均在90%以上，分辨率显著优于其他基因片段。.2）优化了的rbcLb基因区域和ITS引物。.rbcL的适用面广但分辨率低。为了提高rbcL的区分能力和扩增效果，我们提出的rbcLb对被子植物的物种区分度提高了10%。.ITS是目前分辨率最高的来自核基因组的DNA条形码，引物经常扩增出动物和微生物的片段。为了解决这个问题，我们通过计算机模拟和实际检验，设计了生物通用性和植物专一性引物，分子系统学和DNA条形码解决了技术问题。.3）提出了DNA修复技术。我们采用了DNA片段重构技术，克服了利用标本馆材料所遇到的DNA降解问题。.4）重新界定了的李属。广义李属的界定是属于蔷薇科分类的核心问题之一。我们使用了12个叶绿体DNA片段和3个核基因片段，建立了广义李属的系统发育关系，从而提出了广义李属1属3亚属和李亚属7组的分类新体系。这个新分类系统将被应用在《Flora of Pan-Himalaya》中。.5）短片段叶绿体基因组测序策略。我们开展了比较叶绿体基因组学研究，开发了138对被子植物通用引物。应用这些引物，任何科学家都可以测定一般被子植物的叶绿体基因组，使他们迈进了基因组时代。.蔷薇科是个大科，存在广泛的无融合生殖现象，形态变异复杂，研究难道极大。要解决这样一个重要类群的物种问题，需要科学基因的持续资助。